The World Health Organization estimates that one in three human beings is believed to be infected with Mycobacterium tuberculosis (Styblo, K.,Reviews of Infectious Diseases, Vol. II, Suppl. 2, March-April 1989; and Bloom and Murray, Science, 257:1055-1067 (1992)). Over the past decade, there has been a recent resurgence in the incidence of tuberculosis in developed countries that has coincided with the AIDS epidemic (Snider and Roper, N. England J. Med., 326:703-705 (1992)). Because of their impact as major human pathogens and as a result of their profound immunostimulatory properties, mycobacteria have long been intensively studied. In the early 1900s, an attenuated mycobacterium, Mycobacterium (M.) bovis Bacille Calmette-Guerin (M. bovis BCG or BCG), was isolated for use as a vaccine against tuberculosis (Calmette et al., Acad. Natl. Med. (Paris), 91:787-796 (1924); reviewed in Collins, F. M., Bacterial Vaccines (R. Germanier, ed.), Academic Press, pp. 373-418, 1984). Although the efficacy of this vaccine against tuberculosis varied considerably in different trials, and the reasons for its variable efficacy have yet to be resolved, BCG is among the most widely used human vaccines (Luelmo, F., Am. Rev. Respir. Dis., 125:70-72 (1982); and Fine, P. E. M., Reviews of Infectious Diseases II, Supp. 2:5353-5359 (1989)).
The recent application of molecular biological technology to the study of mycobacteria has led to the identification of many of the major antigens that are targets of the immune response to infection by mycobacteria (Kaufmann, S. H. E., Immunol. Today, 11:129-136 (1990); Young, R. A., Ann. Rev. Immunol., 8:401-420 (1990); Young et al., London: Academic Press Ltd., pp. 1-35, 1990; and Young et al., Mol. Microbiol., 6:133-145 (1992)) and to an improved understanding of the molecular mechanisms involved in resistance to antimycobacterial antibiotics (Zhang et al., Nature 358:591-593 (1992); and Telenti et al, Lancet, 341:647-650 (1993). The development of tools that permit molecular genetic manipulation of mycobacteria has also allowed the construction of recombinant BCG vaccine vehicles (Snapper et al., Proc. Natl. Acad. Sci. USA, 85:6987-6991 (1988); Husson et al., J. Bacteriol., 172:519-524 (1990); Martin et al., B. Nature, 345:739-743 (1990); Snapper et al., Mol. Microbiol., 4:1911-1919 (1990); Aldovini and Young, Nature, 351:479-482 (1991); Jacobs et al., Methods Enzymol., 204:537-555 (1991); Lee et al., Proc. Natl. Acad. Sci. USA, 88:3111-3115 (1991); Stover et al., Nature, 351:456-460 (1991); Winter et al., Gene, 109:47-54 (1991); and Donnelly-Wu et al., Mol. Microbiol., 7:407-417 (1993)). Genome mapping and sequencing projects are providing valuable information about the M. tuberculosis and M. leprae genomes that will facilitate further study of the biology of these pathogens (Young and Cole, J. Bacteriol., 175:1-6 (1993)).
Despite these advances, there are two serious limitations to our ability to manipulate these organisms genetically. First, very few mycobacterial genes that can be used as genetic markers have been isolated (Donnelly-Wu et al., Mol. Microbiol., 7:407-417 (1993)). In addition, investigators have failed to obtain homologous recombination in slow growing mycobacteria, such as M. tuberculosis and M. bovis BCG (Kalpana et al., Proc. Natl. Acad. Sci. USA, 88:5433-5447 (1991); and Young and Cole, J. Bacteriol., 175:1-6 (1993)), although homologous recombination has been accomplished in the fast growing Mycobacterium smegmatis (Husson et al., J. Bacteriol., 172:519-524 (1990)).
Described herein is a method of transforming slow-growing mycobacteria, such as M. bovis BCG, M. leprae, M. tuberculosis, M. avium, M. intracellulare and M. africanum; a method of manipulating genomic DNA of slow-growing mycobacteria through homologous recombination; a method of producing homologously recombinant (HR) slow-growing mycobacteria in which heterologous DNA is integrated into the genomic DNA at a homologous locus; homologously recombinant (HR) slow-growing mycobacteria having heterologous DNA integrated into their genomic DNA at a homologous locus; and mycobacterial DNA useful as a genetic marker.
Applicants have succeeded in introducing heterologous DNA (i.e., transforming) into slow-growing mycobacteria through the use of electroporation in water (rather than in buffer). In the present method of transforming slow-growing mycobacteria, heterologous DNA (such as linear DNA or plasmid DNA) and slow-growing myco-bacteria (e.g., M. bovis BCG, M. leprae, M. tuberculosis, M. avium, M. intracellulare and M. africanum) are combined and the resulting combination is subjected to electroporation at an appropriate potential and capacitance for sufficient time for the heterologous DNA to enter the slow growing mycobacteria, resulting in the production of transformed mycobacteria containing the heterologous DNA. In one embodiment, heterologous DNA and M. bovis BCG are combined and subjected to electroporation in water. In a particular embodiment, the M. bovis BCG-heterologous DNA combination is subjected to electroporation in water at settings of approximately 2.5 kV potential and approximately 25 xcexcF capacitance. Optionally, prior to harvest, cells to be transformed are exposed to glycine (such as by adding 1-2% glycine to culture medium in which the slow-grow mycobacteria are growing) in order to enhance or improve transformation efficiencies. In one embodiment, 1.5% glycine is added to the culture medium 24 hours prior to harvesting of the cells, which are then combined with heterologous DNA to be introduced into the slow-growing mycobacteria. The resulting combination is subjected to electroporation, preferably in water, as described above.
In a further embodiment of the method of transforming slow growing mycobacteria, cultures of the cells are maintained in (continuously propagated in) mid-log growth, in order to increase the fraction of cells which are undergoing DNA synthesis (and which, thus, are competent to take up heterologous DNA). Cultures of cells maintained in log-phase growth are subjected to electroporation, preferably in water and, as a result, are transformed with the heterologous DNA. As described above, efficiency of transformation can be increased by exposing the slow-growing mycobacteria to glycine prior to electroporation. Thus, in this embodiment, slow-growing mycobacteria in log-phase growth are combined with heterologous DNA (e.g., plasmid DNA, linearized DNA) to be introduced into the slow-growing mycobacteria. The resulting combination is subjected to electroporation (preferably in water), under conditions (potential and capacitance settings and sufficient time) appropriate for transformation of the cells. Optionally, prior to electroporation, the log-phase cells are exposed to glycine (e.g., approximately 1-2% glycine added to culture medium) in order to enhance transformation efficiency.
Heterologous DNA introduced into slow-growing mycobacteria is DNA from any source other than the recipient mycobacterium. It can be homologous to DNA present in the recipient mycobacterial genomic DNA, nonhomologous or both. DNA which is homologous to mycobacterial genomic DNA is introduced into the genomic DNA by homologous recombination or integration. Alternatively, the heterologous DNA introduced by the present method can be nonhomologous and, thus, enter mycobacterial genomic DNA by random integration events or remain extrachromosomal (unintegrated) after it enters the mycobacterium. In addition, in one embodiment of the present method, nonhomologous DNA linked to or inserted within DNA homologous to genomic DNA of the recipient mycobacterium is introduced into genomic DNA of the recipient mycobacterium as a result of homologous recombination which occurs between genomic DNA and the homologous DNA to which the nonhomologous DNA is linked (or in which it is inserted). For example, as described herein, a mycobacterial gene which encodes a genetic marker has been identified and isolated and used to target homologous integration of heterologous DNA (DNA homologous to genomic DNA of the mycobacterial recipient, alone or in conjunction with DNA not homologous to genomic DNA of recipient mycobacteria) into genomic DNA of a slow-growing mycobacterium. Specifically, the M. bovis BCG gene encoding orotidine-5-monophosphate decarboxylase (OMP DCase) (uraa) has been isolated, as has DNA flanking OMP DCase. The OMP DCase gene and the flanking DNA have been sequenced. The mycobacterial DNA containing the uraA locus, modified to contain heterologous DNA (a selectable marker gene) has been used to carry out integration of the heterologous DNA (the mycobacterial DNA and the selectable marker gene) into mycobacterial genomic DNA, resulting in production of homologously recombinant mycobacteria containing the heterologous DNA of a homologous locus. Specifically, M. bovis BCG DNA containing the uraA locus and flanking sequences was modified to replace the OMP DCase coding sequence with the Kanr selectable marker gene (aph). The resulting construct, which included approximately 1.5 kb uraA flanking sequences on each side of the selectable marker gene, was transformed into M. bovis BCG, using the method described above. M. bovis BCG cultures in mid-log growth were subjected to electroporation in water, resulting in transformation of cells with the construct. Transformants were selected for further study, which showed that all transformants assessed contained vector DNA integrated into the genome and that in some of the transformants, the transforming DNA had integrated at the homologous genomic locus. Thus, heterologous DNA of interest has been introduced into genomic DNA of slow-growing mycobacteria through homologous recombination, to produce homologously recombinant slow-growing mycobacteria in which the heterologous DNA is integrated into the homologous genomic locus (a genomic locus homologous to at least a portion of the heterologous DNA).
Heterologous DNA which includes DNA homologous to genomic DNA of the recipient mycobacterium (homologous DNA) and DNA which is not homologous to genomic DNA of the recipient mycobacterium (nonhomologous DNA) can be introduced into (transformed into) slow growing mycobacterium by the present method for several purposes. As described herein, heterologous nonhomologous DNA encoding a product to be expressed by the resulting homologously recombinant slow-growing mycobacterium has been introduced into mycobacterial genomic DNA at a locus homologous with additional sequences to which the nonhomologous DNA is linked. In this embodiment, the DNA construct transformed into recipient slow-growing myco-bacteria comprises homologous DNA, which directs or targets introduction of the heterologous DNA into the homologous locus of the mycobacterial genome, and nonhomologous DNA, which is expressed in transformed homologously recombinant mycobacteria. In this embodiment, the nonhomologous DNA is introduced into mycobacterial genomic DNA in such a manner that it is added to the genomic DNA or replaces genomic DNA. In a second embodiment, heterologous DNA integrated into genomic DNA is not expressed in the recipient cells. In this embodiment, the DNA construct includes homologous DNA for targeting into a homologous genomic locus and DNA which acts to knock out (inactivate) or activate a resident mycobacterial gene. In the case of inactivation, the mycobacterial gene is xe2x80x9cknocked outxe2x80x9d, in the sense that it is rendered inactive by addition of DNA whose presence interferes with its ability to function, by removal or replacement of sequences necessary for it to be functional or by its complete removal from the mycobacterial genome. In the case of activation, the heterologous DNA integrated into the genomic DNA turns on or enhances expression of a mycobacterial gene, such as by introducing a heterologous promoter which controls the mycobacterial gene expression. In the embodiment in which heterologous DNA affects expression of an endogenous mycobacterial gene, the homologous DNA can serve both functions (i.e., the targeting and inactivation/activating functions); if that is the case, the DNA construct includes only homologous DNA. Alternatively, the DNA construct can include homologous DNA (for targeting purposes) and nonhomologous DNA (for altering function of the mycobacterial gene).
Homologously recombinant slow-growing mycobacteria of the present invention are useful, for example, as vehicles in which proteins encoded by the heterologous nonhomologous DNA are expressed. They are useful as vaccines, which express a polypeptide or a protein of interest (or more than one polypeptide or protein), such as an antigen or antigens of one or more pathogens against which protection is desired (e.g., to prevent or treat a disease or condition caused by the pathogen). Pathogens of interest include viruses, retroviruses, bacteria, mycobacteria, other microorganisms, organisms or substances (e.g., toxins or toxoids) which cause a disease or condition to be prevented, treated or reversed. The homologously recombinant slow-growing bacteria can also be used to express enzymes, immunopotentiators, lymphokines, pharmacologic agents, antitumor agents (e.g., cytokines), or stress proteins (useful for evoking or enhancing an immune response or inducing tolerance in an autoimmune disease). For example, homologously recombinant slow-growing mycobacteria of the present invention can express polypeptides or proteins which are growth inhibitors or are cytocidal for tumor cells (e.g., interferon xcex1, xcex2 or xcex3, interleukins 1-7, tumor necrosis factor (TNF) xcex1 or xcex2) and, thus, are useful for treating certain human cancers (e.g., bladder cancers, melanomas). Homologously recombinant slow-growing mycobacteria of the present invention are also useful vehicles to elicit protective immunity in a host, such as a human or other vertebrate. They can be used to produce humoral antibody immunity, cellular immunity and/or mucosal or secretory immunity. The antigens expressed by the homologously recombinant slow-growing mycobacteria, useful as vaccines or as diagnostic reagents, are also the subject of the present invention. In addition, homologously recombinant slow-growing mycobacteria of the present invention are useful as vaccines in which the heterologous DNA introduced through homologous integration is not itself expressed, but acts to knock out a mycobacterial gene necessary for pathogenicity of the slow-growing mycobacterium or its growth in vivo. Such homologously recombinant slow-growing mycobacteria are useful as vaccines to provide protection against diseases caused by the corresponding wild-type mycobacterium or as a vaccine vehicle which contains a gene(s) encoding an antigen(s) of a different pathogen(s) (e.g., as a vaccine to provide protection against an organism other than the corresponding wild-type mycobacterium or against a toxin or toxoid).
The vaccine of the present invention has important advantages over presently available vaccines. For example, mycobacteria have adjuvant properties; they stimulate a recipient""s immune system to respond to other antigens with great effectiveness. In addition, the mycobacterium stimulates long-term memory or immunity. This means that a single (one time) inoculation can be used to produce long-term sensitization to protein antigens. Long-lasting T cell memory, which stimulates secondary antibody response neutralizing to the infectious agent or toxic. This is particularly useful, for example, against tetanus and diphtheria toxins, pertussis, malaria, influenza, herpes viruses and snake venoms.
BCG in particular has important advantages as a vaccine vehicle. For example, it can be used repeatedly in an individual and has had a very low incidence of adverse effects. In addition, BCG, as well as other mycobacteria, have a large genome (approximately 3xc3x97106 bp in length). As a result, a large amount of heterologous DNA can be accommodated within (incorporated into) the mycobacterial genome, which means that a large gene or multiple genes (e.g., DNA encoding antigens for more than one pathogen) can be inserted into genomic DNA, such as by homologous recombination.